DECONTAMINATION PROTOCOL OPTIMIZATION AND BIOFILM INHIBITION ASSESSMENT ON Cu- AND ZnO-INCORPORATED COMPOSITE RESINS

Orthodontic resin composites modified with copper (Cu) and zinc (Zn) particles aim to prevent biofilm and caries. However, disc decontamination prior to antibiofilm activity tests is critical to avoid baseline contamination and cross-microbial interference, particularly when reusing the discs. Validating effective decontamination protocols without altering material properties is essential for reliable antibiofilm evaluation. This study aimed to establish an effective decontamination protocol for Cu- and Zn-based resin composite discs without altering their properties, and to evaluate their antibiofilm activity against a multispecies biofilm model. Cu- and Zn-based resin composite discs (0.075% Cu, 1% ZnO, 0.075% Cu/1% ZnO) were evaluated. Decontamination protocols evaluated a multi-step treatment starting with an initial mechanical wash with neutral detergent on all discs, followed by detergent immersion under agitation (15 min), detergent sonication (15 min), and isopropyl alcohol sonication (10 min). Additional sterility protocols tested included 70% ethanol immersion under agitation (15 or 20 min) or UV-C light exposure (15 min). Sterility was monitored in 6-well plates with BHI broth at 37 °C, tracking OD_600nm for up to 24h. For biofilm assay, Streptococcus mutans, Lactobacillus casei, Candida albicans were grown in BHI broth for 24h (37 °C, 5% CO₂), transferred to TSB broth with 1% glucose for 18 h, and then to TSB with 1% sucrose under the same conditions. Multispecies suspensions (10⁷, 10⁹, 10⁵ CFU/mL, respectively) were inoculated onto the artificial saliva- conditionate discs for 1 h at 37 °C in 24-well plates. Biofilm formation was quantified by crystal violet absorbance assays. Standard 15-min 70% ethanol treatment proved insufficient for disc decontamination, mainly after reutilized discs, resulting in microbial growth within 24 h. In contrast, the optimized multi-step cleaning protocol completely prevented contamination, maintaining a growth-free medium. Initial growth curves across BHI, MRS, and TSB confirmed robust growth for all species, ensuring their viability in TSB for biofilm assays. Additionally, BHI supported efficient growth for all tested strains, validating its use as a single pre-culturing medium. In conclusion, the multi-step decontamination protocol effectively ensures disc sterility, while standardized microbial growth kinetics establish a reliable foundation for evaluating the antibiofilm activity of Cu- and Zn-based resin discs.